The discovery that crossing two inbred plant lines yields hybrids with increased productivity has generated great interest among plant breeders in the development of economical methods for the production of hybrid seed. Hybrids generally tend to be more productive, and are more efficient in utilization of nutrients, such as fertilizers and water. F.sub.1 hybrids also tend to exhibit superior resistance to environmental stress than parental inbred lines, and can also exhibit desirable disease resistance traits. By producing hybrids of two inbred lines, it may also be possible to combine characteristics that are difficult or impossible to combine in other ways.
Hybrid vigor is a common phenomenon in plants and animals, and has been commercially exploited in plant breeding for many years. Commercial hybrids are presently available for many economically important crop species, and such efforts continue for crops in which hybrids can be produced reliably and economically.
In order to produce hybrid progeny of two inbred lines, cross-pollination must occur. This presents a barrier to hybrid production for many crop plants which are naturally self-pollinating, producing both pollen and egg, often in the same flower. To prevent self-fertilization, the pollen-producing organ must be removed or destroyed in one parent. This may be accomplished by hand-emasculation, i.e., removal of the entire male flower, or removal of anthers from flowers having both functional male and female organs within the same flower. Hand-emasculation is a labor intensive and resultingly expensive process.
Male-sterile parent lines for hybrid seed production may also be produced by the use of chemicals ("gametocides") that prevent formation of viable pollen. These chemicals are also expensive, and are not fully reliable due to limitations in applying the chemicals to the plants.
Uniform cross-pollination may also be accomplished by using genetically male-sterile plants as female parents, planted next to pollen-producing male parents. Using this method, all seed harvested from the row of female parents result from cross-pollination.
Lack of pollen production (i.e., male sterility) in a plant can be due to nuclear mutations or to cytoplasmically-inherited factors. Cytoplasmic male sterility (CMS) is preferred over nuclear-genetic male sterility for the production of hybrid seed because the CMS characteristic is not subject to Mendelian segregation. Hanson & Conde, Int'l. Rev. Cytol., 94: 213-267 (1985). To illustrate, nuclear-genetic male sterility is usually encoded by a single recessive gene, thereby requiring homozygosity for the male-sterile phenotype to be expressed. To propagate nuclear-genetic male sterile plants, the homozygous recessive male steriles must be crossed with an isogenic male-fertile line that is heterozygous for the male sterility gene. Such crosses result in the formation of some percentage of male-fertile plants (50% in a single-gene system), which must be rouged from the field as soon as their fertility can be identified, in order to maintain the effectiveness of the desired male-sterile population. Similar to hand-emasculation, rouging of male-fertile plants from a field is labor intensive and expensive. Thus, segregation of nuclear genes greatly limits the usefulness of nuclear-genetic male sterility for producing hybrid seed.
Nuclear-genetic male sterility suffers from the drawback of Mendelian segregation. By contrast, cytoplasmic male sterility is expressed in all offspring of a hybrid cross between a CMS inbred and a male-fertile parent. It is for this reason that the CMS characteristic is the preferred method for production of hybrid seed.
Naturally-occurring cytoplasmic male sterility is attributable to mutations in the mitochondrial genome. The relationships between these mitochondrial mutations and cytoplasmic male sterility is variable from species to species, and is not well understood. In maize, one of the best-characterized mitochondrial CMS systems, cytoplasmic male sterility in the Texas cytoplasm (CMS-T) appears to be associated with the expression of a 13-kD polypeptide. Hansen, Ann. Rev. Genet., 25:461-486 (1991); Levings & Siedow, Plant Molec. Biol., 19: 135-147 (1992). It was shown that this polypeptide is a transmembrane protein that increases the permeability of cell membranes. The polypeptide has no obvious detrimental effect in normally-dividing vegetative cells, but by an as yet unknown mechanism, causes excessive permeability of tapetal cell membranes and consequent disruption of pollen formation. Production of the 13 kD polypeptide has been correlated with susceptibility of CMS-T maize to plant pathogens such as Bipolaris maydis and Phyllosticta maydis.
In plant breeding programs where naturally-occurring CMS cytoplasm is available, seed production requires three inbred lines: (1) a cytoplasmically male-sterile line having a CMS cytoplasm; (2) a fertile inbred with normal cytoplasm, which is isogenic with the CMS line for nuclear genes ("maintainer line"); and (3) a fertile inbred with normal cytoplasm, carrying a fertility restoring gene ("restorer" line, which need not be isogenic with the CMS line). The CMS line is propagated by pollination with the maintainer line. All plants from this cross will be male-sterile since the CMS cytoplasm is derived from the female parent. Hybrid seed is produced by pollination with a second inbred line carrying fertility restorer (Rf) gene. If no restorer gene is available, sterile hybrids still can be obtained by pollination with a different inbred that does not carry a fertility restorer gene. Such hybrids are useful for crops in which the vegetative tissue is utilized (e.g., tobacco leaf or petunia flowers). However, in most crops, the seeds are the valuable portion of the crop, so fertility of the hybrids in these crops must be restored.
Although cytoplasmic male sterility is useful for hybrid seed production, its usefulness is often limited by the numerous practical problems associated with naturally-occurring cytoplasmic male sterility. These include the inability to identify CMS cytoplasms or nuclear restorer alleles, as well as assorted undesirable traits such as disease sensitivity (as described above), reduced fertility, and the like. Each of these difficulties arises from the lack of understanding of the mitochondrial mechanism for cytoplasmic male sterility and restoration of fertility. Clearly, the value of cytoplasmic male sterility would be greatly increased if a CMS system were developed whose components were fully understood and characterized, and which could be confidently manipulated without the risk of detrimental side effects.